Monday, 9 July 2012

TOPLESS across plant evolution

Although plants and animals became multicellular organisms independently they use many of the same mechanisms to control the complex feat of going from one cell to trillion. Both need to produce organs (arms or leaves) and their embryos need to decide what is top and bottom (head vs feet or shoot vs root). After a round of random mutagenesis (Long et al., 2002; Long et al., 2006), identified a mutant, which at elevated temperatures had the human equivalent of two heads, one of them being where its legs/feet should be. This doubled rooted phenotype was caused by a mutation in a transcriptional co-repressors then named TOPLESS due to the lack of a ‘top’ (shoot) in the mutant plant. The proteins encoded by the TOPLESS gene in plants has related co-repressors in animals. Their job is to regulated which genes are turned off. But they do no bind DNA directly and have no innate specificity for particular genes. Specificity is achieved through transcription factors binding target genes and recruiting co-repressors, which mediates the repression of these genes. It is still not fully understood why the mutation in TOPLESS causes the double rooted phenotype as the TOPLESS protein is involved in many processes including regulating stem cell number and flowering time (Causier et al., 2012a). However, it probably involves the plant hormone auxin. This hormone has a role in nearly every aspect of plant development, including top-bottom (apical-basal) patterning. It has been shown TOPLESS is used by regulators of auxin-dependent gene expression (Aux/IAA proteins) to turn of genes (Szemenyei et al., 2008).

We decided to ask when TOPLESS become involved with auxin during the evolution of plants. The above work was done in the model flowering plant Arabidopsis thaliana. The first flowering plants emerged during the time of the dinosaurs (about 150 million years ago) but plants first appeared on land about half a billion years ago! One of these early groups of land plants resembled modern day mosses. Taking living mosses like the lab model Physcomitrella patens, we can ask if the same mechanisms controlling growth existed in the last common ancestor of mosses and flowering plants, which existed about half a billion years ago.

Using a technique called yeast-two hybrid we can detect if two proteins physically interact. We took the two moss TOPLESS proteins and three Aux/IAA proteins and tested their interaction. As expected, both TOPLESS proteins interacted with all three Aux/IAA proteins (Causier et al., 2012b). Previous work has shown A. thaliana TOPLESS interacts with Aux/IAA proteins by using their LxLxL motif in domain 1 (where L is Leucine and x is any amino acid). Moss TOPLESS proteins have an LxLxPP (P is Proline) instead and might be the functional equivalent (Paponov et al., 2009). We mutated the second L of the LxLxPP motif and found no interaction between TOPLESS proteins and Aux/IAA proteins (Causier et al., 2012b).

Previously, it was shown some auxin response factors (ARF proteins) from A. thaliana interact with TOPLESS (Causier et al., 2012a). These are not the ARFs involved with the ‘classical’ auxin signalling. These are repressors and little is know about them. This work suggests they may act as repressors thanks to the actions of TOPLESS. We investigated if these interactions were also conserved in moss. Although not all repressive ARFs in moss interacted with TOPLESS, two did (Causier et al., 2012b). These are form the ARF10/16/17 family.

Together, these data show that TOPLESS has been involved with auxin signalling components for the last 500 million years since the origins of land plants. Perhaps these interactions pre-date land plants? But we have shown using simple lab techniques we can try to answer fundamental questions about how the regulation of gene expression has evolved.

Actually, I do have a question. Maybe I missed it in the post, but do you also get a double rooted phenotype in moss when you mutate the equivalent of TOPLESS? Is this event possible in moss (I remember you telling me that the genetics of moss are not as advance as in other organisms)

Very interesting post James, good to see the blog is back in action. Is it possible to tell if the interacting ARFs from Arabidopsis and moss are orthologous? If not, do they show any common features which are not found in other ARFs?

So the ARF family has been looked at in plants. Basically 3 clades appear but within each of these there are subclades of just moss or Arabidopsis genes suggesting last time moss and Arabidopsis there were 3 ARFs.

In the large Arabidopsis study my lab did they found ARFs 2, 9, 18 from one clade and 17 from another (which also contains ARFs 10 and 16) interacted with TOPLESS and related proteins. We checked to see if homologues of ARF2/9/18 in moss interacted and they didn't (in Y2H at least) but both of the homologous genes of ARF10/16/17 in moss interacted. I hope that answered your question.

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We are geneticists and biochemists, alumni of the University of York (2009), now doing PhDs at the Universities of Cambridge, Leeds, Oxford and Vermont. We aim to bring to your attention interesting science, whether it is making headlines or not, referencing the original peer-reviewed research as often as possible.